Percutaneous transplantation of pulverized bone in rabbits

The effects of percutaneous injection of the pulverized bone on osteogenesis at the site of bone defect were investigated in rabbits. Although the ceramic bone mixed with blood coagula showed the osteogenic capacity in fresh bone defects, it did not show this capacity in old bone defect. Injection of pulverized bone mixed with blood coagula resulted in greater bone formation than did pulverized bone mixed with saline. Three types of pulverized bone were used: ceramic bone, Kiel bone and autologous corticocancellous bone. Ceramic bone showed the greatest capacity in osteogenesis. These results suggest that the percutaneous injection of pulverized ceramic bone is effective in the treatment of fresh bone defects.     

The extractable calcitonin value in normal individuals]

Human calcitonin levels have been measured by many authors, however the limit of detectability of human calcitonin is as high as 10 pg/ml, and its normal value may range from 10 to 249 pg/ml. To avoid the limitations of present human calcitonin assays, Hunter Heath in 1983 developed a silica Sep-Pap extraction procedure. In this study, extractable calcitonin (Monomeric CT = CT-M) in 261 normal individuals was determined using Heath's method. The CT-M value ranged from 3.0 to 17.4 pg/ml with an average of 7.5 +/- 2.2 pg/ml. The limit of detectability of CT-M was 1.0 pg/ml. The CT-M value was higher in males than in females in all generations, and was highest in the third decade and slightly decreased in the seventh decade. CT-M seemed to accurately reflect human calcitonin in the pure form. The values of human calcitonin determined in this study may be useful in the clinical laboratory.     

A clinicopathological study of ameloblastomas

72 cases of ameloblastoma were obtained from the files of the Department of Pathology, School of Dentistry, Aichi-Gakuin University for the years January 1970 through December 1983. The cases were analyzed with respect to sex, age, duration, site histopathology, and treatment. Of 72 patients, 63 had no previous therapy, while 9 received their initial treatment elsewhere. There were 38 males and 34 females, a ratio of 1.2: 1. At the time of diagnosis, the ages of all patients ranged from 11 to 71 years, with an average of 36.6 years. About 65% of patients were in the 2nd, 3rd, and 4th decades of life. The duration of symptoms varied from 2 days to 5 years, with an average of 12.6 months. 69 cases occurred in the mandible, with the molar-ramus region being the most frequent site of involvement. Only 3 were found in the maxilla. The left side of the mandible was affected 1.6 times more frequently than the right. Histopathologically, 44 cases were of the plexiform type, 15 the follicular, 10 the acanthomatous, and 2 the basal cell type. Only 1 case was of the granular cell type. Most of the findings in the present study agreed with previous available data from the literature on ameloblastomas.     

Ultrastructural observation on Langerhans cells in the rat gingival epithelium

The present study was carried out to investigate ultrastructurally Langerhans cells in the rat gingival epithelium. The gingivae of lower incisors of 15 Wistar rats were examined by electron microscopy. The results were as follows: Langerhans cells were observed mainly in the lower prickle-cell layer of the gingival epithelium. On rare occasions Langerhans cells were also found in the basal and granular layers. The average number of Langerhans cells per 100 cells in the prickle-cell layer was 1.0 cell. Usually Langerhans cells had clear cytoplasms and convoluted or indented nuclei, although sometimes the cells exhibited round nuclei. The clear cytoplasm contained a moderately developed Golgi apparatus and a small number of rough surfaced endoplasmic reticulum, free ribosomes, mitochondria, and lysosomes, but it lacked tonofilaments. Birbeck granules were often found in close vicinity of the Golgi apparatus. The average number of Birbeck granules per one Langerhans cell was 4.3 granules. The cell membrane of Langerhans cells had no junctional complexes like desmosomes. The degeneration of keratinocytes adjacent to Langerhans cells was observed in a few specimens.     

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

Proton-coupled electron transfer (PCET), a ubiquitous phenomenon in biological systems, plays an essential role in copper nitrite reductase (CuNiR), the key metalloenzyme in microbial denitrification of the global nitrogen cycle. Analyses of the nitrite reduction mechanism in CuNiR with conventional synchrotron radiation crystallography (SRX) have been faced with difficulties, because X-ray photoreduction changes the native structures of metal centers and the enzyme–substrate complex. Using serial femtosecond crystallography (SFX), we determined the intact structures of CuNiR in the resting state and the nitrite complex (NC) state at 2.03- and 1.60-Å resolution, respectively. Furthermore, the SRX NC structure representing a transient state in the catalytic cycle was determined at 1.30-Å resolution. Comparison between SRX and SFX structures revealed that photoreduction changes the coordination manner of the substrate and that catalytically important His255 can switch hydrogen bond partners between the backbone carbonyl oxygen of nearby Glu279 and the side-chain hydroxyl group of Thr280. These findings, which SRX has failed to uncover, propose a redox-coupled proton switch for PCET. This concept can explain how proton transfer to the substrate is involved in intramolecular electron transfer and why substrate binding accelerates PCET. Our study demonstrates the potential of SFX as a powerful tool to study redox processes in metalloenzymes.Since the invention of the Haber–Bosch process, the amount of fixed nitrogen in soils and waters has been increasing, and this trend has significant impact on the global environment (1, 2). Fixed nitrogen is oxidized to nitrite (NO₂⁻) or nitrate (NO₃⁻) by nitrification and then converted to gaseous dinitrogen (N₂) by microbial denitrification, which closes the nitrogen cycle. Microorganisms involved in denitrification couple their respiratory systems to stepwise reduction of nitrogen oxides to N₂ (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (3, 4). The reduction of NO₂⁻ to toxic nitric oxide (NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O) is referred to as the key step in denitrification and catalyzed by either cd₁-heme nitrite reductase (cd₁NiR) or copper nitrite reductase (CuNiR) (3, 4). Although the catalytic mechanism of cd₁NiR is well understood (5, 6), that of CuNiR is controversial (7). CuNiR is a homotrimeric protein containing two distinct Cu sites per monomer (SI Appendix, Fig. S1). Type 1 Cu (T1Cu) with a Cys–Met–His₂ ligand set is an electron acceptor incorporated near the molecular surface, whereas type 2 Cu (T2Cu) with a His₃ ligand set is a catalytic center, which is ∼12 Å distant from the molecular surface and located between two adjacent monomers (7, 8). Spaced ∼12.5 Å apart, the two Cu sites are linked by a Cys–His bridge and a sensor loop. Whereas the Cys–His bridge is an electron pathway, the sensor loop is thought to control electron distribution between T1Cu and T2Cu (9).Two conserved residues, Asp98 and His255 (Alcaligenes faecalis numbering), are located above the T2Cu site and bridged by a water molecule called bridging water (SI Appendix, Fig. S1). They are essential to the CuNiR activity because they assist proton transfer (PT) to the substrate (10–12). Although intramolecular electron transfer (ET) from T1Cu to T2Cu can occur in the resting state (RS) (13, 14), the differences in the redox potentials of T2Cu minus T1Cu are small and sometimes negative in the absence of NO₂⁻, meaning that intramolecular ET before NO₂⁻ binding is not energetically favorable (15, 16). By contrast, intramolecular ET is dramatically accelerated in the presence of NO₂⁻ (15, 17). An explanation for this gating-like phenomenon is that substrate binding raises the redox potential of T2Cu and shifts the equilibrium of the ET reaction (16). However, pH dependence of intramolecular ET in the presence of NO₂⁻ cannot be explained by such a change of redox potentials (15). Instead, Kobayashi et al. (15) proposed that reduction-induced structural change of His255 is responsible for the gating-like mechanism. Because it has been recently proven that intramolecular ET in CuNiR is accompanied by PT and hence proton-coupled ET (PCET) (17, 18), one can readily speculate that intramolecular ET contributes PT to NO₂⁻ and that the structural change of His255 is involved in PCET. Crystal structures of CuNiR from Rhodobacter sphaeroides (RhsNiR) implies this possibility because His287 in RhsNiR, which corresponds to His255, seems to show pH- and redox-dependent conformational changes (19, 20). However, presumably because of X-ray radiation damages implied by rerefinement of RhsNiR structures (21), electron density around His287 was so unusual that interpretation of it is difficult (SI Appendix, Fig. S2).Crystal structures determined by synchrotron radiation crystallography (SRX) have provided insights into the enzymatic mechanism of CuNiR (22–25), and these studies are summarized elsewhere (7). High-resolution nitrite complex (NC) structures revealed an O-coordination of NO₂⁻ showing a near face-on binding mode (22, 23), whereas Cu(II)-NO₂⁻ model complexes show a vertical binding mode (7, 26–29). The near face-on coordination manner is thought to facilitate its conversion to side-on NO, which was observed in the crystal structures of CuNiR exposed to NO (22, 23, 25). Skeptical eyes have, however, been cast on these CuNiR structures because SRX data might be affected by some problems connected to the high radiation dose delivered on the crystals. First, strong synchrotron X-rays cause not only radiation damages to amino acid residues but also photoreduction of metalloproteins (30, 31). Although a comparison between oxidized and reduced states is necessary to closely investigate redox reactions, completely oxidized structures are almost impossible to determine by SRX. Indeed, the Cu centers in CuNiR are rapidly reduced by exposure to synchrotron X-rays (21, 32). Second, following the photoreduction of T2Cu, NO₂⁻ is easily reduced and produces NO and water in SRX (21). Consequently, electron density at the catalytic site of an NC structure is derived from the mixture of both substrate and product, making interpretation of data complicated and unreliable. Third, cryogenic manipulations for reducing radiation damages in SRX have also been focused as a factor that changes the population of amino acid residues (33, 34) and enzyme–substrate complexes (35). Crystallographic (36), computational (37), and spectroscopic (38–40) studies actually show that binding modes of NO₂⁻ and NO in CuNiR crystal structures can differ from those in physiological environments.We here ventured to use photoreduction in SRX to initiate a chemical reaction and to trap an enzymatically produced intermediary state (30, 31). Furthermore, to visualize intact CuNiR structures in the resting and NC states, we applied serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) (41), which enables damage-free structural determination of metalloproteins (42, 43) and evaluation of the native conformational population at room temperature (RT) (44). By comparing SRX and SFX data, we discuss PCET and nitrite reduction in CuNiR.